A biophysical study on how the actin and microtubule cytoskeletons dynamically collaborate to regulate cellular organization

A fundamental question in biology is how complex animals originate from the first cell, the egg. Embryologists suggested long ago that the asymmetric distribution of substances, called determinants, in the cytoplasm of an egg (known as egg polarization) could confer a specific characteristic to the cells that receive them upon cell division. One of the most striking examples of egg polarization is found in Drosophila, in which the asymmetric localization of various determinants during oogenesis establishes the body plan. For these developmental determinants to localize properly, a variety of complex processes need to take place. Firstly, cells (such as the egg) are not symmetric entities; rather they have to adopt particular asymmetries, which are critical to define the final position of the determinants. Additionally, the fluid through which the determinants navigate, known as cytoplasm is not quiescent. Instead it is a swirling, jetting, dynamic fluid. Thus, to understand how the asymmetric distribution of substances is achieved, we need to study not only the movement of the substance, but also the motion of the highly dynamic cytoplasm that surrounds the substance.

The link between the movement of molecules and the fluid dynamics of the cytoplasm is not only a theoretical one, but also a mechanistic one. Cells have two complex filamentous structures, known as the actin and microtubule (MT) cytoskeletons that exert forces and drive transport of molecules and organelles. These cytoskeletons, and a group of proteins (known as molecular motors) that "walk" along them, are required to transport determinants, but also to induce cytoplasmic flows. At times actin, MTs and their motor proteins act as separate systems, but there is also often poorly understood crosstalk between the two cytoskeletons. In the Drosophila female germline, localization of developmental determinants is governed by the interplay among actin, MTs and the motor protein Kinesin. Both directed transport and cytoplasm streaming, are mediated by this interplay. This constitutes a powerful cellular organization model of developmental importance in which the two cytoskeletons dynamically modulate each other.

We will employ a unified theoretical and experimental approach to understand how the actin and MT cytoskeletons coordinate the essential, and developmentally regulated, asymmetries and motions during oogenesis. Further, we will explore how streaming impacts on the organization of the cytoskeletons, and what flows reveal about the underlying forces. Specifically, we will investigate: 1. The bi-directional link between cytoplasmic flows and the MT cytoskeleton; and 2. The regulation of streaming, MTs and Kinesin by the actin cytoskeleton.

Cellular asymmetries strongly rely on actin and microtubule (MT) cytoskeletons. At times these act as separate systems, but there is often poorly understood crosstalk between the two cytoskeletons. During Drosophila midoogenesis, developmental determinants are transported on MTs. The action of the motor Kinesin on MTs also induces cytoplasmic streaming. It is hypothesized that cytoplasmic F-actin regulates both processes, but the nature of the crosstalk between actin and microtubules and the interplay between streaming and the cytoskeletons remain largely unknown. Flows, actin and MTs not only interact during mid-oogenesis, when the oocyte is highly polarized, but also in late oogenesis, when the cell undergoes a rapid reorganization. The project focuses on using biophysics to study the bi-directional interaction between the cytoplasm fluid dynamics and the cytoskeletons, using tools of quantitative imaging, theoretical fluid dynamics, microfluidics and genetics. We believe the oocyte to be a powerful cellular organization model in which the two cytoskeletons dynamically modulate each other: How the two cytoskeletons interact with each other in regulating cellular organization is an area of increasing importance.

Our aims are to: i) Study the relation between motors, viscosity and flows. In vivo work will be complemented by in vitro assays to test the biophysical relation between the force generating entities and flows; ii) Analyze the correlation between flows and two movements of the MTs: the bulk arrangements of bundles, and the dynamics of the plus-ends; iii) Analyze whether the actin mesh regulates flows, kinesin and MT organization. We will also characterize the biophysical features of the actin mesh, and how they correlate with those of streaming.

This proposal fits well into the priority 'systems approach to biological research' by using experimental and theoretical physics to study a complex system determined by the properties of many moving parts

This proposal will contribute to the understanding of basic cellular mechanisms that are essential for cell organization and cell asymmetries. This contribution is also important for human health for several reasons. Since the cytoskeleton is involved in virtually all cellular processes, including cell proliferation, abnormalities in this cellular component frequently result in disease. Also, there is some evidence that the defective function of motors results in various human diseases, such as Alzheimer and retinitis pigmentosa. Kinesin is specifically linked to Charcot-Marie-Tooth disease and Hereditary Spastic Paraplegia. In addition, the asymmetric localization of molecules is important not only in the establishment of the mammalian body plan, but also in the function of most cells, including neurons, where it is involved in learning and is defective in various diseases. The application explores a fundamental problem at the interface between biology and physics, and thus has a potential transformative capability, which affect many fields. Also, this approach may help create a new field of biophysics, so its long-term impact could be very high. The strong contribution of this research to medicine would have an positive effect on the competitiveness of the economy and would lead to improvement of our quality of life.

Our research will also generate benchmark observations, which will connect with growing interest within the fluid dynamics community in the behavior of systems composed of many motile components. A considerable amount of recent research in this general area suggested interesting potential applications in autonomous robotics and microfluidics.

Exploitation: Although this proposal mainly contributes to the understanding of basic cellular mechanisms, it is possible in an unpredictive manner that it leads to new ways of preventing abnormal cell behavior and human diseases (e.g., through the development of new drugs). Other areas for possible exploitation include the field of microfluidics, where mixing on the micron scale is a longstanding challenge. If this were the case, the University of Cambridge has extensive experience of technology transfer through their company Cambridge Enterprise

Outreach: Our results will be presented at a wide range of interdisciplinary scientific meetings worldwide, and at various EU summer schools/workshops, which Goldstein co-directs in the areas of biological physics. Both principal investigators are linked to various outreach associations. These are: The Cambridgeshire Branch of the British Science Association; The Cambridge Association for Women in Science and Engineering (AWiSE), The Millennium Maths Project, and public lectures through the Institute of Physics and the Royal Institution. In addition, both PIs plan to contribute in the next few years to the Cambridge Science Festival (Goldstein has previously participated), one of the largest in the country http://comms.group.cam.ac.uk/sciencefestival/. As a former URF, Palacios is in continuous contact with the Royal Society, which influences policymaking with its scientific advice and invigorates science education. The Department of Applied Mathematics and Theoretical Physics regularly has Open Days in which hundreds of members of the public view a wide range of research activities in areas as diverse as fluid dynamics, particle physics, and cosmology

Skills:There are various skills that the staff working on the project will develop which they could apply to other sectors. These are: project management; definition and achievement of milestones; efficiently working within a group; successfully deal with deadlines; designing/performing/analyzing experiments;communication skills both in writing and in oral presentations to worldwide peers; and commercial awareness. The fundamental interdisciplinary character of the research provides the opportunity to improve communication skills among a very diverse group of scientists